Signal transduction occurs in complex and redundant protein interaction networks, and multi-protein complexes are characterized by dynamic changes in their component interactions. Therefore, these processes are best understood by simultaneously monitoring the localizations and activities of multiple proteins under near-physiological conditions. Forster resonance energy transfer (FRET) microscopy with fluorescent protein (FP)-based biosensors can dynamically image changes in protein interactions or conformation. However, FRET biosensor imaging is hampered by the broad absorption and emission spectra of FPs and high levels of autofluorescence background that lower signal-to-noise ratio (S/N), lower detection limits (requiring high levels of protein expression), necessitat multiple control images that limit temporal resolution (3-5 s image acquisition), and severely hinder the ability to perform multiplexed imaging of 2 or more FRET pairs. The goal of this project is to deliver 10-fold improvements in the temporal resolution and sensitivity of live-cell, multiplexed biosensor imaging via the use of lanthanide probes and time-gated microscopy. The objectives are to i) synthesize dendritic lanthanide (Tb, Eu) protein labels with 10-fold greater brightness than existing probes; ii) directly compare lanthanide-based and conventional FRET imaging using model dual- and single- chain biosensors; iii) image multiple dynamic interactions between the epithelial tight junction protein ZO-1 and claudin2, occludin and F-actin that are linked to disease-related changes in epithelial barrier function. Our central hypothesis is that biosensors containing multiple lanthanide complex donors will increase FRET signals proportionally with the lanthanide donor/acceptor ratio. This hypothesis rests on data from the previous cycle showing that i) conjugation to cell penetrating peptides (CPPs) mediates consistent cytoplasmic delivery of protein-targeted, ligand-Tb complex heterodimers and specific, stable labeling of receptor fusion proteins; and ii) both intermolecular and intramolecular Tb-to-GFP FRET can be detected in single-channel, time-gated images at cellular protein levels (1-10 uM), image acquisition times (1-3 s) and image S/N (>5) that are comparable to those seen with current, intensity-based FP-FRET imaging methods. The rationale for this project is that the development and quantitative evaluation of next-generation, lanthanide FRET biosensors will have a significant positive impact on human health by enabling 10X more potential protein interactions or activities that can be successfully imaged with minimal cell perturbation and commensurately greater capability of integrating dynamic data into static models of protein networks.